Abstract
The emergence of super-resolution (SR) fluorescence microscopy has rejuvenated the search for new cellular sub-structures. However, SR fluorescence microscopy achieves high contrast at the expense of a holistic view of the interacting partners and surrounding environment. Thus, we developed SR fluorescence-assisted diffraction computational tomography (SR-FACT), which combines label-free three-dimensional optical diffraction tomography (ODT) with two-dimensional fluorescence Hessian structured illumination microscopy. The ODT module is capable of resolving the mitochondria, lipid droplets, the nuclear membrane, chromosomes, the tubular endoplasmic reticulum, and lysosomes. Using dual-mode correlated live-cell imaging for a prolonged period of time, we observed novel subcellular structures named dark-vacuole bodies, the majority of which originate from densely populated perinuclear regions, and intensively interact with organelles such as the mitochondria and the nuclear membrane before ultimately collapsing into the plasma membrane. This work demonstrates the unique capabilities of SR-FACT, which suggests its wide applicability in cell biology in general.
Highlights
In terms of the search for new structures and dynamics, the emergence of super-resolution (SR) fluorescence microscopy techniques in the 21st century is expected to reshape all aspects of modern life science[1]
To increase the temporal resolution while measuring the scattered optical field of light passing through biological samples due to the inhomogeneous distribution of the refractive index (RI), we built an off-axis holographic optical diffraction tomography (ODT) system based on a commercial microscope (IX73, Olympus) equipped with a galvo-mirror scanning mechanism
The 561nm light was divided into two beams by a polarizationdependent beam splitter (CCM1-PBS251, Thorlabs), of which one beam was used to illuminate the sample on the microscope stage, while the other served as a reference
Summary
In terms of the search for new structures and dynamics, the emergence of super-resolution (SR) fluorescence microscopy techniques in the 21st century is expected to reshape all aspects of modern life science[1]. Because different cellular organelles exhibit different refractive index (RI) values[3], they may cause the scattering of incident light in live cells, which can be measured by phase-contrast microscopy techniques such as differential interference contrast microscopy[4]. The image contrast and resolution of fast-moving structures such as lysosomes will be compromised in live-cell experiments (detailed in Supplementary Notes 1.1, Supplementary Fig. S1). A lack of molecular specificity in label-free ODT microscopy hinders the interpretation of imaging results. Two-dimensional phase-contrast microscopy can be combined with fluorescent confocal microscopy for livecell studies, the low resolution of label-free images prevents the accurate prediction of the organelles within cells[11,12]. All previous dual-mode imaging microscopy techniques failed when used for time-lapsed correlated SR imaging in live cells[11,12,13], which is the focus of this study
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
